Unilateral Ureteral Occlusion Research Articles

ELABELA-derived peptide ELA13 attenuates kidney fibrosis by inhibiting the Smad and ERK signaling pathways.

Kidney fibrosis is an inevitable result of various chronic kidney diseases (CKDs) and significantly contributes to end-stage renal failure. Currently, there is no specific treatment available for renal fibrosis. ELA13 (amino acid sequence: RRCMPLHSRVPFP) is a conserved region of ELABELA in all vertebrates; however, its biological activity has been very little studied. In the present study, we evaluated the therapeutic effect of ELA13 on transforming growth factor-β1 (TGF-β1)-treated NRK-52E cells and unilateral ureteral occlusion (UUO) mice. Our results demonstrated that ELA13 could improve renal function by reducing creatinine and urea nitrogen content in serum, and reduce the expression of fibrosis biomarkers confirmed by Masson staining, immunohistochemistry, real-time polymerase chain reaction (RT-PCR), and western blot. Inflammation biomarkers were increased after UUO and decreased by administration of ELA13. Furthermore, we found that the levels of essential molecules in the mothers against decapentaplegic (Smad) and extracellular signal-regulated kinase (ERK) pathways were reduced by ELA13 treatment in vivo and in vitro. In conclusion, ELA13 protected against kidney fibrosis through inhibiting the Smad and ERK signaling pathways and could thus be a promising candidate for anti-renal fibrosis treatment.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction against renal fibrosis.

To investigate the mechanism by which Sini decoction (, SND) improves renal fibrosis (Rf) in rats based on transforming growth factor β1/Smad (TGF-β1/Smad) signaling pathway. Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf. The targets of SND drug components and the targets of Rf were obtained by searching databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSMP) and GeenCard. The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram. Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-β1/Smad signaling pathway. The Rf rat model was established by unilateral ureteral occlusion (UUO). The expression levels of transforming growth factor, matrix metalloproteinase-9 (MMP-9), matrix metal protease-2 (MMP-2), connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by Masson staining of rat renal tissue, and immunohistochemical methods. The expression levels of Smad3, Smad2, and Smad7 in renal tissue were determined by Western blotting (WB). The mechanism of the improving effects of SND on Rf was investigated based on TGF-β1/Smad signaling pathway. A total of 12 drug components of Fuzi (Radix Aconiti Lateralis Preparata), 5 drug components of Ganjiang (Rhizoma Zingiber), and 9 drug components of Gancao (Radix Glycy et Rhizoma) were obtained from the database search, and 207 shared targets were found. A total of 1063 Rf targets were found in the database search. According to the Venn diagram, in total, 96 intersection targets were found in two database searches. The metabolic pathways involved included TGF-β signaling pathway, phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling (PI3K/Akt) pathway, and hypoxia-inducible factor-1 (HIF-1) signaling pathway. Masson staining analysis showed that compared with the model group, the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower (P < 0.05). Immunohistochemical analysis, compared with the control group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 were significantly increased (P < 0.01). Compared with the model group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 in the kidney tissue were significantly decreased (P < 0.05, P < 0.01). WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3 (P < 0.05) and increase the expression of Smad7 (P < 0.05).

RAS protein activator-like 2 (RASAL2) initiates peritubular capillary rarefaction in hypoxic renal interstitial fibrosis

The progression of chronic kidney disease (CKD) often involves renal interstitial fibrosis (RIF) and subsequent loss of peritubular capillaries (PTCs), which enhances disease severity. Despite advancements in our understanding of fibrosis, effective interventions for reversing capillary loss remain elusive. Notably, RIF exhibits reduced capillary density, whereas renal cell carcinoma (RCC) shows robust angiogenesis under hypoxic conditions. Using RNA sequencing and bioinformatics, we identified differentially expressed genes (DEGs) in hypoxic human renal tubular epithelial cells (HK-2) and renal cancer cells (786-0). Analysis of altered Ras and PI3K/Akt pathways coupled with hub gene investigation revealed RAS protein activator-like 2 (RASAL2) as a key candidate. Subsequent in vitro and in vivo studies confirmed RASAL2′s early-stage response in RIF, which reduced with fibrosis progression. RASAL2 suppression in HK-2 cells enhanced angiogenesis, as evidenced by increased proliferation, migration, and branching of human umbilical vein endothelial cells (HUVECs) co-cultured with HK-2 cells. In mice, RASAL2 knockdown improved Vascular endothelial growth factor A (VEGFA) and Proliferating cell nuclear antigen (PCNA) levels in unilateral ureteral occlusion (UUO)-induced fibrosis (compared to wild type). Hypoxia-inducible factor 1 alpha (HIF-1α) emerged as a pivotal mediator, substantiated by chromatin immunoprecipitation (ChIP) sequencing, with its induction linked to activation. Hypoxia increased the production of RASAL2-enriched extracellular vesicles (EVs) derived from tubular cells, which were internalized by endothelial cells, contributing to the exacerbation of PTC loss. These findings underscore RASAL2′s role in mediating reduced angiogenesis in RIF and reveal a novel EV-mediated communication between hypoxic tubular- and endothelial cells, demonstrating a complex interplay between angiogenesis and fibrosis in CKD pathogenesis.

Fibroblast activation protein-sensitive polymeric nanobeacon for early diagnosis of renal fibrosis

Early diagnosis and treatment of renal fibrosis (RF) significantly affect the clinical outcomes of chronic kidney diseases (CKDs). As the typical fibrotic ailment, RF is characterized by remodeling of the extracellular matrix, and the activation of fibroblast activation protein (FAP) plays a crucial role in the mediation of extracellular matrix protein degradation. Therefore, FAP can serve as a biomarker for RF. However, up to now, no effective tools have been reported to diagnose early-stage RF via detecting FAP. In this work, a polymeric nanobeacon integrating an FAP-sensitive amphiphilic polymer and fluorophores was proposed, which was used to diagnose early RF by sensing FAP. The FAP can be detected in the range of 0 to 200 ng/mL with a detection limit of 0.132 ng/mL. Furthermore, the fluorescence imaging results demonstrate that the polymeric nanobeacon can sensitively image fibrotic kidneys in mice with unilateral ureteral occlusion (UUO), suggesting its potential for early RF diagnosis and guidance of FAP-targeted treatments. Importantly, when employed alongside with non-invasive diagnostic techniques like magnetic resonance imaging (MRI) and serological tests, this nanobeacon exhibits excellent biocompatibility, low biological toxicity, and sustained imaging capabilities, making it a suitable fluorescent tool for diagnosing various FAP-related fibrotic conditions. To our knowledge, this study represents the first attempt to image RF in early stage by detecting FAP, offering a promising fluorescent molecular tool for diagnosing various FAP-associated diseases in the future.

Inhibition of PFKP in renal tubular epithelial cell restrains TGF-β induced glycolysis and renal fibrosis

Metabolic reprogramming to glycolysis is closely associated with the development of chronic kidney disease (CKD). Although it has been reported that phosphofructokinase 1 (PFK) is a rate-limiting enzyme in glycolysis, the role of the platelet isoform of PFK (PFKP) in kidney fibrosis initiation and progression is as yet poorly understood. Here, we investigated whether PFKP could mediate the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells (PTECs). We induced PFKP overexpression or knockdown in renal tubules via an adeno-associated virus (AAV) vector in the kidneys of mice following unilateral ureteral occlusion. Our results show that the dilated tubules, the area of interstitial fibrosis, and renal glycolysis were promoted by proximal tubule-specific overexpression of PFKP, and repressed by knockdown of PFKP. Furthermore, knockdown of PFKP expression restrained, while PFKP overexpression promoted TGF-β1-induced glycolysis in the human PTECs line. Mechanistically, Chip-qPCR revealed that TGF-β1 recruited the small mothers against decapentaplegic (SMAD) family member 3-SP1 complex to the PFKP promoter to enhance its expression. Treatment of mice with isorhamnetin notably ameliorated PTEC-elevated glycolysis and kidney fibrosis. Hence, our results suggest that PFKP mediates the progression of kidney interstitial fibrosis by regulating glycolysis in PTECs.

MircroRNA-92b as a negative regulator of the TGF-β signaling by targeting the type I receptor

Transforming growth factor β1 (TGFβ1) has been identified as a major pathogenic factor underlying the development of chronic kidney disease (CKD). This study investigated the role of miR-92b-3p in the progression of renal fibrosis in unilateral ureteral occlusion (UUO) and unilateral ischemia-reperfusion injury (uIRI) mouse models, as well as explored its underlying mechanisms in human proximal tubular epithelial (HK2) cells. We found that renal fibrosis increased in UUO mice after miR-92b knockout, while it reduced in miR-92b overexpressing mice. MiR-92b knockout aggravated renal fibrosis in uIRI mice. RNA-sequencing analysis, the luciferase reporter assay, qPCR analysis, and western blotting confirmed that miR-92b-3p directly targeted TGF-β receptor 1, thereby ameliorating renal fibrosis by suppressing the TGF-β signaling pathway. Furthermore, we found that TGF-β suppressed miR-92b transcription through Snail family transcriptional repressors 1 and 2. Our results suggest that miR-92b-3p may serve as a novel therapeutic for mitigating fibrosis in CKD.

RGS1 mediates renal interstitial fibrosis through activation of the inflammatory response

Renal interstitial fibrosis (RIF) is considered as a common pathway for all patients with chronic kidney disease (CKD) to progress to end-stage kidney disease (ESRD). The basic pathological manifestation is the increase of matrix component in the tubular interstitium, while the injury of tubular epithelial cells in the renal interstitium and the excessive accumulation of matrix will eventually lead to tubular atrophy and obstruction, loss of effective renal units, and finally impaired renal filtration function. The relevant mechanism of RIF remains unclear. The present study will investigate the function and relevant mechanism of RGS1 in RIF. The RIF-related microarrays GSE22459 and GSE76882 were downloaded and analyzed. Renal parenchymal atrophic calyx tissues were collected from clinical RIF patients. Cellular inflammation, fibrosis and animal RIF models were constructed using Lipopolysaccharide (LPS), TGF-β1 and unilateral ureteral occlusion (UUO). HE and Masson staining were performed to detect morphological alterations of renal tissue samples. qRT-PCR, Western blot and ELISA were carried out to detect the expression of relevant genes/proteins. RGS1 is a gene co-differentially expressed by GSE22459 and GSE76882. RGS1 expression was elevated in renal tissues of RIF patients, cells and animal RIF models. Knockdown of RGS1 inhibited renal cell inflammatory response, fibrosis and renal fibrosis in RIF mice. Overexpression of RGS1 plays the opposite role. Knockdown of RGS1 inhibited the inflammatory response in the RIF cell and mouse model. Targeting RGS1 might be a potential therapeutic strategy for RIF treatment.

#4966 CELLULAR PRION PROTEIN ACTIVATES THE TBK1-IRF3 SIGNALING PATHWAY TO AGGRAVATE RENAL FIBROSIS

Abstract Background and Aims Chronic kidney disease (CKD) is an irreversible degenerative disease characterized by gradual loss of renal function, contributing to high morbidity and mortality as well as heavy economic cost to society. Clinically, there are few available strategies to slow CKD progression. Because the uremic phenotypes include many features of aging, CKD is considered as a premature aging syndrome. Neurodegenerative diseases, such as Alzheimer's disease, and Parkinson's disease are extensively studied aging-related diseases, in which cytotoxic proteins misfolding and aggregation in neural cells is the main characteristic. The misfolding and aggregation of the prion protein is tightly linked to the development of prion diseases. Interestingly, a remarkable elevation of cellular prion protein (PrPC) in the urine and plasma of CKD patients was reported. However, the role of PrPC in the pathogenesis of kidney diseases remains largely unknown. The aim of the current study is to investigate the role of PrPC in renal fibrosis. Methods Immunohistochemistry staining was conducted to evaluate the expression of PrPC in renal biopsies from CKD patients. Enzyme linked immunosorbent assay (ELISA) was performed to detect urinary PrPC of CKD patients. Animal model of renal fibrosis was induced by unilateral ureteral occlusion (UUO). Western blot, real time PCR and immunofluorescence staining were performed to evaluate the expression and distribution of PrPC. Proximal tubule specific Prnp knockout mice (PEPCK-Prnp-KO) was constructed to explore the pathogenic role of PrPC in renal fibrosis. Primary proximal tubular epithelial cells (PTCs) overexpressed PrPC were collected for RNA-seq analysis. TBK1 inhibitor amlexanox, and IRF3 siRNA were used to block TBK1-IRF3 pathway in rat tubular epithelial cells (NRK-52E), respectively. Amlexanox was administered orally to UUO mice. H&amp;E, Masson and Sirius red staining were used to estimate renal pathology. Results The protein expression levels of PrPC both in renal biopsies and urine from CKD patients were elevated and significantly correlated with the severity of interstitial fibrosis and the decline of eGFR. PrPC proteins were significantly increased, and aggregated in proximal renal tubules in fibrotic renal tissues induced by UUO. Compared with wild-type littermates, PEPCK-Prnp-KO mice showed reduced extracellular matrix accumulation and interstitial inflammation at day 7 after UUO. PrPC provokes profibrotic response of renal tubular cells via the TBK1-IRF3 pathway. TBK1 inhibitor amlexanox or silencing endogenous IRF3 significantly inhibited PrPC-induced profibrogenic phenotypic transformation in NRK-52 cells. Amlexanox attenuated interstitial fibrosis induced by UUO. Conclusion Our experimental results indicate that PrPC-TBK1-IRF3 pathway plays a detrimental role in profibrogenic transformation of renal tubular cells and thus contributes to interstitial fibrosis. Blocking TBK1 activation is a plausible strategy for therapeutic intervention of chronic kidney disorders.

MicroRNA-26a-5p Restoration Ameliorates Unilateral Ureteral Obstruction-Induced Renal Fibrosis in Mice Through Modulating TGF-β Signaling

Renal fibrosis is a hallmark of chronic and progressive renal diseases characterized by excessive fibroblast proliferation, extracellular matrix accumulation, and a loss of renal function, eventually leading to end-stage renal diseases. MicroRNA-26a-5p (miR-26a-5p) downregulation has been previously noted in the sera of unilateral ureteral occlusion (UUO)–injured mice, and exosome-mediated miR-26a-5p reportedly attenuated experimental pulmonary and cardiac fibrosis. This study evaluated the expression patterns of miR-26a in a human tissue microarray with kidney fibrosis and in tissues from a mouse model of UUO-induced renal fibrosis. Histologic analyses showed that miR-26a-5p was downregulated in human and mouse tissues with renal interstitial nephritis and fibrosis. Moreover, miR-26a-5p restoration by intravenous injection of a mimic agent prominently suppressed the expression of transforming growth factor β1 (TGF-β1) and its cognate receptors, the inflammatory transcription factor NF-κB, epithelial-mesenchymal transition, and inflammatory markers in UUO-injured kidney tissues. In vitro, miR-26a-5p mimic delivery significantly inhibited TGF-β1−induced activation of cultured normal rat kidney NRK-49F cells, in terms of downregulation of TGF-β1 receptors, restoration of the epithelial marker E-cadherin, and suppression of mesenchymal markers, including vimentin, fibronectin, and α-smooth muscle actin, as well as TGF-β1/SMAD3 signaling activity. Our findings identified miR-26a-5p downregulation in kidney tissues with human interstitial nephritis and UUO-induced mouse kidney fibrosis. MiR-26a-5p restoration may exhibit an antifibrotic effect through the blockade of both TGF-β and NF-κB signaling axes and is considered a novel therapeutic target for treating obstruction-induced renal fibrosis.

M6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis.

The dysregulation of autophagy contributes to renal fibrosis. N6-Methyladenosine (m6A) RNA modification is a critical mediator of autophagy. Our previous studies have reported that the disorder of the PPARα/fatty acid oxidation (FAO) axis in renal tubular cells is suppressed by STAT6, which is involved in the regulation of renal fibrotic processes. Here, we found that canagliflozin significantly upregulates SQSTM1/P62, promoting PPARα-mediated FAO by inducing autophagy-dependent STAT6 degradation both in TGF-β1-treated HK2 cells and in unilateral ureteral occlusion (UUO) and ischemia-reperfusion (I/R) renal fibrosis mouse models. Knockdown of P62/SQSTM1 led to the impairment autophagic flux and the dysregulation of the STAT6/PPARα axis, which was confirmed by SQSTM1/P62cKO mice with UUO treatment along with bioinformatics analysis. Furthermore, SQSTM1/P62 deficiency in renal tubular cells inhibited canagliflozin's effects that prevent FAO disorder in renal tubular cells and renal fibrosis. Mechanistically, the level of m6A eraser FTO, which interacted with SQSTM1 mRNA, decreased in the renal tubular cells both in vitro and in vivo after canagliflozin administration. Decrease in FTO stabilized SQSTM1 mRNA, which induced autophagosome formation. Collectively, this study uncovered a previously unrecognized function of canagliflozin in FTO in the autophagy modulation through the regulation of SQSTM1 mRNA stability in the renal tubular STAT6/PPARα/FAO axis and renal fibrosis.

Chrysophanol, a main anthraquinone from Rheum palmatum L. (rhubarb), protects against renal fibrosis by suppressing NKD2/NF-κB pathway

PurposeChronic kidney disease (CKD), characterized as renal dysfunction and multi-system damage, has become a serious public health problem with high prevalence and mortality. Rheum palmatum L. (rhubarb) is one of the most widely used Chinese herb with renal protective activity. However, the active components and underlying mechanisms of rhubarb remain unknown. In this work, we tried to explore the pharmacological mechanism of chrysophanol, a main anthraquinone from rhubarb, against CKD by in vivo and in vitro models. Study designThe therapeutic effect of chrysophanol and its underlying mechanism were investigated using CKD mouse model induced by unilateral ureteral occlusion (UUO), and human kidney 2 (HK-2) cells stimulated by TGF-β1 in vivo. MethodsThe impact of chrysophanol on renal function, inflammation, fibrosis of CKD mice were evaluated. Then, the protein expressions of FN1, collagen ɑI, α-SMA, NF-κB and naked keratinocyte homolog 2 (NKD2) were investigated. In vitro studies, the inhibition on inflammation and fibrogenesis by chrysophanol was further validated in TGF-β1-stimulated HK2 cells, and the regulation of chrysophanol on NKD2/NF-κB pathway was analyzed. Moreover, NKD2 was overexpressed in HK-2 cells to confirm the role of NKD2/NF-κB pathway in chrysophanol-mediated efficacy. Finally, the binding mode of chrysophanol with NKD2 was studied using in silico molecular docking and microscale thermophoresis (MST) assay. ResultsChrysophanol could significantly improve the kidney dysfunction, alleviate renal pathology, and reverse the elevated levels of renal fibrosis markers such as FN1, collagen ɑI and α-SMA. Furthermore, chrysophanol effectively inhibited TNF-α, IL-6, and IL-1β production, and suppressed NF-κB activation and NKD2 expression. The findings of in vitro study were consistent with those of animal expriment. Using NKD2-overexpressing HK-2 cells, we also demonstrated that overexpression of NKD2 significantly compromised the anti-fibrotic effects of chrysophanol. In addition, molecular docking and MST analysis revealed that NKD2 was a direct target of chrysophanol. ConclusionTogether, our work demonstrated for the first time that chrysophanol could effectively ameliorate renal fibrosis by inhibiting NKD2/NF-κB pathway. Chrysophanol can potentially prevent CKD by suppressing renal NKD2 expression directly.

Targeted inhibition of ZAK ameliorates renal interstitial fibrosis

ZAK (sterile alpha motif and leucine zipper-containing kinase) is a newly discovered member of the subfamily of mitogen-activated protein kinase kinase kinases (MAP3Ks). The role of ZAK in kidney disease remains largely unknown. In this study, we systematically investigated the expression and function of ZAK in the progression of tubulointerstitial fibrosis (TIF). ZAK was induced, predominantly in tubular epithelium, in both fibrotic kidneys of human and mouse models with TIF. ZAK expression level was correlated with the extent of renal fibrosis and the decline of eGFR of CKD patients. Depleting ZAK attenuated TIF and inflammation induced by unilateral ureteral occlusion (UUO) together with decreased activation of p38 MAPK and Smads signaling. Moreover, we demonstrated that overexpressed ZAK was in complex with Smad2/3 and TGF-β receptor Ⅰ (TβRI). Whereas, silencing endogenous ZAK ameliorated the amount of Smad2/3 recruited to TβRI. Moreover, we discovered a novel small molecule inhibitor of ZAK, named 6p. In vitro, incubation with 6p inhibited TGF-β1-induced fibrogenic response in NRK52E cells. In vivo, intragastric administration of 6p ameliorated TIF and inflammation in UUO and unilateral ischemia-reperfusion injury model. Delayed administration of 6p was also effective in retarding the progression of the established TIF. In conclusion, ZAK is a novel therapeutic target for TIF, and 6p might be a potential therapeutic agent for TIF.

MicroRNA miR-4709-3p targets Large Tumor Suppressor Kinase 2 (LATS2) and induces obstructive renal fibrosis through Hippo signaling

ABSTRACT Obstructive renal fibrosis is the consequence of abnormal extracellular matrix assembly, which eventually results in renal failure, acute, and end‑stage renal infection. MicroRNAs (miRNAs), a particular category of small RNAs, modulate the expression of genes post-transcriptionally and regulate biological activities, including fibrogenesis. The study probed to estimate the key functions of miR-4709-3p in obstructive renal fibrosis. This investigation used TGF-β1 stimulated HK-2 in-vitro model, unilateral ureteral occlusion (UUO) mice model, and human Diabetic nephropathy (DN) and Renal interstitial fibrosis (RIF) specimens to depict the abundance of the miR-4709-3p level using FISH and RT-qPCR. MiR-4709-3p mimics and inhibitors were utilized to evaluate the functions of miR-4709-3p in-vitro. Luciferase assay was exploited to verify miR-4709-3p and LATS2 3ʹUTR binding. Finally, to depict the functions of miR-4709-3p in-vivo, the UUO model was injected with miR-4709-3p inhibitors. Results exhibited the upregulation of miR-4709-3p in UUO-induced in-vivo model, TGF-β1 stimulated HK-2, and human RIF and DN samples. Moreover, it was determined that modulating miR-4709-3p regulated the level of fibrosis markers. Luciferase assay miR-4709-3p modulates renal fibrosis by targeting LATS2. Finally, it was found that miR-4709-3p regulates obstructive renal fibrosis through the Hippo signaling pathway. Overall, the study concludes that aberrant miR-4709-3p expression plays an essential function in the renal fibrosis progression, and miR-4709-3p overexpression could advance obstructive renal fibrosis via LATS2 targeting in Hippo signaling pathway. Therefore, miR-4709-3p inhibition may be a potential renal fibrosis therapy target.

Bone marrow mesenchymal stem cell-derived exosomes improve renal fibrosis by reducing the polarisation of M1 and M2 macrophages through the activation of EP2 receptors.

Renal fibrosis is the pathological outcome of most end‐stage renal diseases, yet there are still limited therapeutic options for it. In recent years, bone marrow mesenchymal stem cell‐derived exosomes (BM‐MSCs) have received much attention. Here, we investigate the therapeutic effect of BM‐MSCs on unilateral ureteral occlusion (UUO)‐induced interstitial fibrosis in the kidney by modulating prostaglandin E2 receptor 2 (EP2). Renal pathological changes were evident in the UUO group compared to the control group, with significantly increased expression of α‐smooth muscle actin (α‐SMA), fibronectin, Ep2 and F4/80+CD86+ and F4/80+CD206+ cells in the UUO group (p< 0.05). Pathological changes were alleviated and F4/80+CD86+ and F480/+CD206+ cells were reduced after exosome or EP2 agonist intervention compared to the UUO group. These data were further confirmed in vitro. Compared to the lipopolysaccharide (LPS) group and the LPS + exosome + Ah6809 group, the lipopolysaccharide (LPS) + exosome group and the LPS + butaprost group showed a significant decrease in α‐SMA expression, a decrease in the number of F4/80+CD86+ and F4/80+CD206+ cells, a decrease in interleukin (IL)‐6 and an increase in IL‐10 levels. Therefore, we conclude that BM‐MSCs can reduce the polarization of M1 and M2 macrophages by activating EP2 receptors, thereby ameliorating renal fibrosis.

Total C-21 Steroidal Glycosides From Baishouwu Ameliorate Hepatic and Renal Fibrosis by Regulating IL-1β/MyD88 Inflammation Signaling.

Fibrosis is a worldwide public health problem, which typically results from chronic diseases and often leads to organ malfunction. Chronic inflammation has been suggested to be the major trigger for fibrogenesis, yet mechanisms by which inflammatory signals drive fibrogenesis have not been fully elucidated. Total C-21 steroidal glycosides (TCSG) from Baishouwu are the main active components of the root of Cynanchum auriculatum Royle ex Wight, which exert hepatoprotective and anti-inflammation properties. In this study, we established a mouse model with the coexistence of hepatic and renal fibrosis and aimed to investigate the effects of TCSG from Baishouwu on fibrosis and explored the potential mechanisms. The results of biochemical and pathological examinations showed that TCSG from Baishouwu improved liver and kidney function and alleviated hepatic and renal fibrosis by reducing collagen and extracellular matrix deposition in bile duct ligation and unilateral ureteral occlusion (BDL&UUO) mice. According to network pharmacology analysis, the mechanisms underlying the effects of TCSG from Baishouwu on hepatic and renal fibrosis were associated with inflammatory response pathways, including “Signaling by interleukins”, “MAP kinase activation”, “MyD88 cascade initiated on plasma membrane”, and “Interleukin-1 family signaling”. Regression analysis and western blot results revealed that IL-1β/MyD88 inflammation signaling played an essential role in the anti-fibrotic effects of TCSG from Baishouwu. Further data displayed that TCSG from Baishouwu affected inflammatory response and extracellular matrix deposition via suppressing the activation of p38 MAPK/JNK and NF-κB p65 signaling cascades both in the liver and kidney of BDL&UUO mice. Thus, our findings suggest TCSG from Baishouwu as a natural regimen against hepatic and renal fibrosis and provide direct evidence that IL-1β/MyD88 signaling crucially contributes to hepatic and renal fibrosis and modulates liver-kidney crosstalk by maintaining tight control over inflammatory responses.

The interaction of S100A16 and GRP78 actives endoplasmic reticulum stress-mediated through the IRE1\u03b1/XBP1 pathway in renal tubulointerstitial fibrosis

Recent studies have indicated that the development of acute and chronic kidney disease including renal fibrosis is associated with endoplasmic reticulum (ER) stress. S100 calcium-binding protein 16 (S100A16) as a novel member of the S100 family is involved in kidney disease; however, few studies have examined fibrotic kidneys for a relationship between S100A16 and ER stress. In our previous study, we identified GRP78 as a protein partner of S100A16 in HK-2 cells. Here, we confirmed a physical interaction between GRP78 and S100A16 in HK-2 cells and a markedly increased expression of GRP78 in the kidneys of unilateral ureteral occlusion mice. S100A16 overexpression in HK-2 cells by infection with Lenti-S100A16 also induced upregulation of ER stress markers, including GRP78, p-IRE1α, and XBP1s. Immunofluorescence staining demonstrated that the interaction between S100A16 and GRP78 predominantly occurred in the ER of control HK-2 cells. By contrast, HK-2 cells overexpressing S100A16 showed colocalization of S100A16 and GRP78 mainly in the cytoplasm. Pretreatment with BAPTA-AM, a calcium chelator, blunted the upregulation of renal fibrosis genes and ER stress markers induced by S100A16 overexpression in HK-2 cells and suppressed the cytoplasmic colocalization of GRP78 and S100A16. Co-immunoprecipitation studies suggested a competitive binding between S100A16 and IRE1α with GRP78 in HK-2 cells. Taken together, our findings demonstrate a significant increase in S100A16 expression in the cytoplasm following renal injury. GRP78 then moves into the cytoplasm and binds with S100A16 to promote the release of IRE1α. The subsequent phosphorylation of IRE1α then leads to XBP1 splicing that activates ER stress.

Gypenoside XLIX loaded nanoparticles targeting therapy for renal fibrosis and its mechanism

Renal fibrosis is the main pathological feature of the occurrence and development of chronic nephropathy. At present, there is no effective treatment, except for renal transplantation and dialysis. Previous studies have shown that nano-preparations can be used as a therapeutic tool to target organs. In this study, we studied the therapeutic effect and mechanism of Chinese medicine monomer Gypenoside (Gyp) XLIX on renal fibrosis and explored the targeting and therapeutic effects of polylactic acid-co-glycoside (PLGA)-Gyp XLIX nanoparticles in unilateral ureteral occlusion (UUO) kidney. Gyp XLIX and PLGA-Gyp XLIX nanoparticles were used to treat UUO mice and Human renal tubular epithelial (HK2) cells stimulated by transforming growth factor-β (TGF-β). Histopathological and molecular biological techniques were used to detect the expression of type I collagen and alpha-smooth muscle actin (α-SMA). To investigate the in vivo targeting of PLGA nanoparticles, they were loaded with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide and injected into UUO mice. We evaluated the effect of Gyp XLIX nanoparticles on TGF-β/Smad3 pathway, a central driver for renal fibrosis in Smad-deficient HK2 cells. Fluorescence imaging showed that the PLGA nanoparticles around 120 nm could be targeted to the UUO kidney. Compared with Gyp XLIX, PLGA-Gyp XLIX nanoparticles could effectively inhibit renal fibrosis and reduce collagen deposition and reduce renal tubular necrosis. Gyp XLIX decreased the phosphorylation of Smad3, but could not further reduce the levels of type I collagen and α-SMA in Smad-deficient cells. This study opens a promising way for targeted drug treatment of renal fibrosis.

Fluorofenidone protects liver against inflammation and fibrosis by blocking the activation of NF-κB pathway.

Despite the increasing understanding of the pathophysiology of hepatic fibrosis, the therapies to combat it remain inadequate. Fluorofenidone (AKF-PD) is a novel pyridone agent able to ameliorate hepatic fibrosis in an experimental hepatic fibrosis model induced by dimethylnitrosamine. However, the underlying mechanism remains to be further elucidated. In light of the critical role of the NF-κB pathway in inflammation and hepatic fibrosis, together with the preliminary finding that AKF-PD decreases the release of proinflammatory cytokines in the endotoxemia and unilateral ureteral occlusion model, the aim of this study was to explore whether AKF-PD exerts an antifibrotic effect in hepatic fibrosis by inhibiting inflammation and suppressing the activation of the NF-κB pathway in vivo and in vitro. To test this possibility, the effect of AKF-PD on hepatic fibrosis models induced by both carbon tetrachloride (CCL4 ) and porcine serum (PS) was investigated. Our results showed that AKF-PD treatment ameliorated hepatic injury and fibrosis in both models. Furthermore, the administration of AKF-PD induced a robust anti-inflammatory reaction revealed by the downregulation of the proinflammatory cytokines as well as the suppression of the infiltration of inflammatory cells in the fibrotic liver. The analysis of the mechanism of action demonstrated that the attenuation of the production of proinflammatory cytokines and chemokines mediated by AKF-PD in vivo and in vitro were accompanied by the suppression in the activation of the NF-κB signaling pathway. In conclusion, AKF-PD might be considered as an antifibrotic agent attenuating hepatic inflammation and fibrosis potentially through the suppression of the NF-κB pathway.

Inhibition of transforming growth factor β1 signaling in resident interstitial cells attenuates profibrotic gene expression and preserves erythropoietin production during experimental kidney fibrosis in mice

Kidney fibrosis is characterized by the development of myofibroblasts originating from resident kidney and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various cytokines. Among these, transforming growth factor β1 (TGFβ1) is considered a central trigger for kidney fibrosis. We found a highly upregulated expression of TGFβ1 and TGFβ receptor 2 (TGFβ-R2) mRNAs in kidney interstitial cells in experimental fibrosis. Here, we investigated the contribution of TGFβ1 signaling in resident kidney interstitial cells to organ fibrosis using the models of adenine induced nephropathy and unilateral ureteral occlusion in mice. For this purpose TGFβ1 signaling was interrupted by inducible deletion of the TGFβ-R2 gene in interstitial cells expressing the fibroblast marker platelet derived growth factor receptor-β. Expression of profibrotic genes was attenuated up to 50% in kidneys lacking TGFβ-R2 in cells positive for platelet derived growth factor receptor-β. Additionally, deletion of TGFβ-R2 prevented the decline of erythropoietin production in ureter ligated kidneys. Notably, fibrosis associated expression of α-smooth muscle actin as a myofibroblast marker and deposits of extracellular collagens were not altered in mice with targeted deletion of TGFβ-R2. Thus, our findings suggest an enhancing effect of TGFβ1 signaling in resident interstitial cells that contributes to profibrotic gene expression and the downregulation of erythropoietin production, but not to the development of myofibroblasts during kidney fibrosis.

Genistein Ameliorates Renal Fibrosis Through Regulation Snail via m6A RNA Demethylase ALKBH5.

Renal tubule-interstitial fibrosis is related to chronic kidney disease progression and a typical feature of the aging kidney. Epigenetic modifications of fibrosis-prone genes regulate the development of renal fibrosis. As a kind of “epigenetic diet”, soy isoflavone genistein was reported to have renal protective action and epigenetic-modulating effects. However, its renal protection role and underlying mechanisms are yet to be fully clarified. Herein, we showed that genistein exhibits a demonstrable anti-fibrotic effect on kidney in vivo UUO (unilateral ureteral occlusion) model and renal epithelial cells in vitro model. The mechanism is strongly associated with epithelial-to-mesenchymal transition and m6A RNA demethylase ALKBH5. Mouse fibrotic kidneys induced by UUO exhibited adverse expression of renal fibrosis-related proteins and significant increases in the total m6A level. As an eraser, ALKBH5 showed severer suppression in the renal fibrosis process. However, genistein pretreatment restored ALKBH5 loss remarkably and reduced renal fibrosis, abnormal protein, and inflammatory markers. The examination of possible mechanisms revealed that genistein promoted ALKBH5 and maybe induced the level of mRNA m6A methylation in some epithelial-to-mesenchymal transition-related transcription factors. We found snail was the critical regulator and critical for the protective role of genistein. To verify the relationship between ALKBH5 and snail, we generated knockdown and overexpression of ALKBH5 cells in vitro. ALKBH5 knockdown enhanced the mesenchymal phenotype marker α-smooth muscle actin and snail expression. In agreement, overexpression ALKBH5 increased epithelial adhesion molecule E-cadherin and reduced snail expression. In conclusion, genistein increased renal ALKBH5 expression in UUO-induced renal fibrosis and reduced RNA m6A levels and ameliorates renal damages.